Several different types of vaccine delivery systems have been described in the literature (see e g “Vaccine Design. The subunit and adjuvant approach” (Eds: M F Powell and M J Newman), Pharmaceutical Biotechnology, vol 6, Plenum Press, NY 1995). Examples of known delivery systems for vaccines include liposomes, cochleates and polymer particles of a biodegradable or non-biodegradable nature. Antigens have also been associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind.
Polymer particles are well suited as vaccine delivery systems, because they can be produced in a range of sizes (eg, microparticles and nanoparticles) according, for example, to the preferred administration route for the vaccine and can slowly release the antigen inside the patient in order to build up a desirable immune response of the patient without the need for multiple vaccinations. The antigen is incorporated with the particles by encapsulation within a matrix of the polymer, with or without adsorption of the antigen onto the surface of the polymer particles.
When the antigen is a protein, care must be taken to chose a preparation method for the polymer particles that does not remove the desired immunogenicity of the protein (eg, by denaturation). Thus, although various techniques are known for generally producing polymer particles with an active drug or substance, as explained below not all of these are well suited to use with a protein antigen.
The following general techniques have been used for preparing polymer particles:                1. Hot Melt Microencapsulation (A. J. Schwope et al Life Sci. 1975, 17,1877);        2. Interfacial Polymerisation (G. Birrenbach & P. Speiser, J. Pharm.Sci. 1976, 65, 1763, Thies, In Encyclopaedia of Chemical Technology, 4 ed., Ed. Kirk-Othmer, 1996, 16, p. 632);        3. Double Emulsion Solvent Evaporation Technique (“Vaccine Design. The subunit and adjuvant approach” (Eds: M F Powell and M J Newman), Pharmaceutical Biotechnology, vol 6, Plenum Press, NY 1995);        4. Double Emulsion Solvent Extraction Technique (“Vaccine Design. The subunit and adjuvant approach” (Eds: M F Powell and M J Newman), Pharmaceutical Biotechnology, vol 6, Plenum Press, NY 1995); and        5. Spray Drying (J. Cox, et al. WO 94/15636).        
In the Hot Melt Microencapsulation method the matrix polymer is melted by heating while mixed with the active substance to incorporated with the particles. This technique is not well suited to use with a proteinaceous active substance, such as protein antigen, since the heating step tends to denature the active substance.
Interfacial Polymerisation is performed in following manner. A core material and the active substance are dissolved in a water immiscible solvent, together with a highly reactive monomer. This solution is then emulsified in water, where another monomer is dissolved, and a stable O/W emulsion is formed. An initiator is added to the water phase and polymerisation occurs, thereby forming a polymer particle incorporating the active substance. When the active substance is proteinaceous, the highly reactive monomer in the water immiscible solvent tends to react undesirably with the active substance as well as the core material, which means that Interfacial Polymerisation is not well suited to the incorporation of a protein antigen with polymer particles.
Techniques are available which entail the formation of a water-in-oil (W/O) emulsion in which the active substance is dissolved in the W phase can be used to incorporate a proteinaceous active substance with polymer particles. Examples are the Double Emulsion Solvent Extraction and Evaporation Techniques and the Spray Drying.
For incorporation of a protein using the Double Emulsion (W/O/X) Solvent Evaporation Technique, a multiple W/O/X emulsion is used. The first step is the formation of a first (W/O) emulsion, in which the protein is dissolved in a first aqueous phase (W) and the oil (O) phase contains the matrix polymer and an organic solvent, the W and O phases being emulsified for example by ultra-sonication. In a second step, this first emulsion is then emulsified in a third phase (X) to form multiple W/O emulsion droplets dispersed in the X is phase, which is commonly a second aqueous phase, but may for example be oil (eg sesame oil) instead. The organic solvent diffuses out from the droplets into the X phase before evaporating from the X phase. Thus, the organic solvent moves from the oil phase of the W/O emulsion droplets, to the X phase and then to the air. This results in a decrease of the organic solvent concentration in the O phase, and opposite an increase in the polymer concentration, since the polymer does not move with the organic solvent to the X phase. At a certain polymer concentration the polymer precipitates, thereby producing polymer particles comprising a matrix of the polymer incorporated with the protein (ie, protein is encapsulated within the matrix with or without surface adsorption onto the outside of the particle).
The Double Emulsion (W/O/X) Solvent Extraction Technique is similar to the Double Emulsion Solvent Evaporation Technique, but the organic solvent is extracted from the O phase of the W/O emulsion instead of being removed by evaporation. In addition, a second oil phase is used as the X phase in the double emulsion. The second oil phase extracts the organic solvent from the O phase, thereby raising the matrix polymer concentration in the O phase and leading to polymer particle formation in which the protein is incorporated with the particles. (Lewis, Drugs and the Pharmaceutical Sciences (M Chasin and R Langer, eds.), Vol. 45, Dekker, New York, 1990, pp 1-42).
In the Spray Drying Technique, a W/O emulsion is formed as discussed above. The emulsion is sprayed through a nozzle to produce small droplets of the emulsion (dispersed in air) from which the solvent rapidly evaporates, thereby leading to formation of polymer particles incorporated with the protein. Microparticles in the 1-10 μm size range can be prepared (at relatively low cost) with this technique.
Biodegradable polymer particles are particularly well suited for use as vaccine delivery systems, because the polymer matrix itself is non-immunogenic and the encapsulation of the antigen protects it from degradation in the gastrointestinal tract (eg, by acid and proteases). An example of an especially suitable matrix polymer is PLG (poly(lactide-co-glycolide) copolymers—also known as PLGA and PLA). PLG particles have excellent tissue biocompatibility, biodegradability and regulatory approval. PLG particles degrade in vivo to form the non-toxic monomers, lactic- and glycolic acids and the release rate of incorporated active substances can be controlled by varying the molecular weight and copolymer ratio.
Examples of documents disclosing the use of Double Emulsion Techniques for incorporating water soluble proteins or peptides with PLG particles include:
H Rafati et al, “Protein-loaded poly(DL-lactide-co-glycolide) microparticles for oral administration: formulation, structural and release characteristics”, J. Controlled Release 43 (1997), pp 89-102. This article discloses the use of a Double Emulsion (W1/O/W2) Solvent Evaporation Technique for incorporating bovine serum albumin (BSA) with particles of PLG.
M J Blanco-Prieto et al, “Characterization and morphological analysis of a cholecystokinin derivative peptide-loaded poly(lactide-co-glycolide) microspheres prepared by a water-in-oil-in-water emulsion solvent evaporation method”, J. Controlled Release 43 (1997), pp 81-87. This article discloses the incorporation of a small water soluble peptide with PLG particles. The authors observe that the stabilisation of the inner emulsion in the double emulsion by the combined use of OVA (ovalbumin) together with the use of a pH gradient between the inner and outer aqueous phase improved peptide encapsulation.
R V Diaz et al, “Effect of surfactant agents on the release of 125I-bovine calcitonin from PLGA microspheres: in vitro—in vivo study”, J. Controlled Release 43 (1997), pp 59-64. This article aims to investigate the possible influence that the surfactants Tween®-80 and Span®-60 (included in the W1 and O phases respectively) could have on the in vitro and in vivo release profile of 125I-bovine calcitonin from PLGA microspheres. The article concludes that the protein encapsulation efficiency is similar independent of the presence or absence of the surfactants.
The prior art has therefore only concerned the incorporation of water soluble proteins and peptides with polymer particles using techniques which involve the formation of a W/O emulsion. The reason for this is that for the desired protein incorporation to take place, the protein must be solubilised in the W aqueous phase in order eventually to produce droplets of W/O emulsion in which the aqueous phase containing the solubilised protein provides the core of the droplets surrounded by the O phase which contains the matrix polymer in an organic solvent.
These techniques have not previously been considered to be useable for the incorporation of water insoluble proteins, because it was thought that these proteins cannot be suitably solubilised in the aqueous W phase.
Note that protein denaturation (eg, unfolding) by the organic solvent precludes the provision of the protein in an O phase together with the matrix polymer in order to produce polymer particles incorporated with a protein antigen. WO 95/11009, WO 95/11010, WO 96/36317, U.S. Pat. No. 5,075,109, U.S. Pat. No. 4,919,929 and U.S. Pat. No. 5,529,777 disclose the formation of microparticles incorporating water soluble antigens. None of these documents discloses the incorporation of a water insoluble protein antigen into polymer particles: WO 95/11009 and WO 95/11010 disclose the microencapsulation of MN rpg120 or QS21 into PLGA; WO 96/36317 discloses the formation of microparticles comprising a polymer matrix (eg, PLG) and a biological agent, further agents being optionally included in order to maintain the potency of the biological agent over the duration of the biological agent's release from the microparticles and to modify the release rate of the biological agent from the microparticles; U.S. Pat. No. 5,075,109 discloses the formation of microparticles incorporating trinitrophenyl keyhole limpet hemocyanin or staphylococcal enterotoxin B as an antigen; and U.S. Pat. No. 5,529,777 discloses the formation of microparticles by mixing a water soluble antigen with a solution of a water soluble polymer or hydrogel. U.S. Pat. No. 5,622,649 discloses W/O and W/O/W emulsions, but there is no disclosure of forming polymer particles. It is essential for the invention disclosed in U.S. Pat. No. 5,622,649 that no hydrophilic surfactant is present in the inner W phase. U.S. Pat. No. 5,622,649 does not disclose a water insoluble protein antigen in the inner W phase.